J. Avian Biol.

39: 647
652, 2008

doi: 10.1111/j.1600-048X.2008.04450.x
# 2008 The Authors. J. Compilation # 2008 J. Avian Biol.
Received 10 December 2007, accepted 11 March 2008

Between-male variation in sperm size, velocity and longevity in sand

martins Riparia riparia

Fabrice Helfenstein, Tibor Szép, Zsolt Nagy, Bart Kempenaers and Richard H. Wagner
F. Helfenstein (correspondence), Evolutionary Ecology Group, Zoological Institute, University of Bern, Baltzerstrasse 6, CH-3012 Bern,
Switzerland. E-mail: fabrice.helfenstein@free.fr.
R. H. Wagner, Konrad Lorenz Institute, Austrian Academy of Sciences, Savoyenstrasse 1a,
A-1160 Vienna, Austria.
T. Sze´p, Institute of Environmental Sciences, College of Nyı´regyha´za, P.O. Box 166, H-4401 Nyı´regyha´za,
Hungary.
Z. Nagy, MME/BirdLife Hungary, Ko¨lto ut 21, H-1121 Budapest, Hungary.
B. Kempenaers, Department Behavioural Ecology,
Evolutionary Genetics, Max Planck Institute for Ornithology, Postfach 1564, D-82305 Starnberg, Germany.

Sperm mobility is known to be an important determinant of a male’s sperm competitive ability. Although more debated,
sperm length and its relation to sperm swimming ability has also been proposed to determine a male’s fertilisation
potential. Furthermore, both mobility and length may covary with a male’s phenotype, either positively (the phenotype-
linked fertility hypothesis) or negatively if, for instance, low-quality males have less access to females but invest more in
sperm production. Using dummy females, we collected sperm samples from wild sand martins Riparia raparia males. We
investigated the relationship between sperm length and sperm swimming speed as measured by sperm straight line
velocity (VSL), and determined whether sperm traits are correlated with male body size and condition. We found that
total sperm length is repeatable within-ejaculate and shows substantial inter-male variation. Sperm length was associated
with sperm velocity: males with short sperm have sperm that swim initially faster but die sooner, whereas males with
longer sperm have sperm that swim more slowly but for a longer time. Smaller males produced sperm with higher overall
velocity. This correlation between male size and sperm behaviour may reflect alternative fertilisation strategies where
small males having less mating opportunities invest more in sperm competitive ability. The existence of such alternative
strategies would participate in maintaining variation in sperm length and velocity in this species.

It is now well established that sperm swimming ability
influences the outcome of sperm competition. In the sea
urchin Lytechinus variegatus, males producing faster sperm
have higher rates of fertilisation (Levitan 2000), while in
boars Sus scrofa domesticus, a variety of sperm motility
parameters are correlated with male fertility (Holt et al.
1997). In birds, sperm mobility or velocity are primary
determinants of male fertility (Froman et al. 1999), and
males having highly mobile sperm sire a greater propor-
tion of offspring in competitive fertilisation experiments
(Birkhead et al. 1999, Donoghue et al. 1999, Denk et al.
2005). Similar results have been found in fish such as the
Atlantic salmon Salmo salar (Gage et al. 2004).
Besides sperm swimming ability, sperm length has been
hypothesized to also play a role in determining a male’s
sperm competitive ability. The exact mechanism by which
this would operate is unknown but it has been proposed
that sperm length is positively related to sperm speed and
thus that sperm length should increase with increasing risk
of sperm competition (Parker 1998). Comparative studies
found some support for this hypothesis in various taxa
including insects, frogs, birds and mammals (Gomendio
and Roldan 1991, 1993, Briskie and Montgomerie 1992,
Gage 1994, Briskie et al. 1997, Byrne et al. 2003; but see
Stockley et al. 1997). Within species some empirical data
also suggest a positive relationship between sperm size and
sperm competitiveness (LaMunyon and Ward 1998,
Oppliger et al. 2003). However, the positive relationship
between sperm length and sperm swimming speed proposed
by theoretical models has seldom been tested (LaMunyon
and Ward 1998, Gage et al. 2004, Birkhead et al. 2005).
Thus, the degree to which sperm size determines success in
sperm competition is currently under debate.
The idea that male phenotype, i.e. social status, body size
or the size of sexual ornaments, may reflect sperm quality is
also under debate. The phenotype-linked fertility hypoth-
esis proposes that females would benefit from choosing
males with elaborated sexual ornaments if they maximize
the chances of mating with fertile males (Sheldon 1994).
Tests of this hypothesis in birds, fish and mammals have
yielded contradictory results with some studies finding a
relationship between male phenotype and ejaculate quality
(Matthews et al. 1997, Simmons and Kotiaho 2002, Peters
et al. 2004, Malo et al. 2005, Schulte-Hostedde and
Montgomerie 2006) while others do not (Birkhead and
Fletcher 1995, Liljedal et al. 1999, Pilastro and Bisazza
1999). Theoretical developments (Parker 1998) also contra-
dict the phenotype-linked fertility hypothesis in suggesting

647
that males that have low access to females, e.g. small males,
may invest more in sperm expenditure to compensate for a
lower number of mating opportunities. Support for this
hypothesis comes from the feral fowl Gallus gallus domes-
ticus where sub-dominant males, which are less preferred by
females and have less access to them, produce more mobile
sperm than dominant males (Pizzari et al. 2007).
Overall, studies testing the relationship between sperm
size and motility and the relationship between male
phenotype and sperm quality are scarce. This is undoubt-
edly because obtaining samples of live sperm from free-
ranging animals is difficult. The goal of our study was to
investigate how sperm velocity, a measure of sperm
swimming ability, sperm length and sperm longevity are
related to each other in the sand martin Riparia riparia. The
sand martin is a colonial, socially monogamous species
where extra-pair copulations and fertilizations are common
and sperm competition is intense (Nicholls et al. 2001).
Sand martin males are known to be prone to copulate with
a dummy female, thus allowing the collection of natural
ejaculates (Nicholls 2000). To test whether male phenotype
may signal sperm characteristics in the sand martin, we
examined correlations between both sperm length and
velocity and body measures and condition. As intra-
ejaculate variation in sperm morphology may be indicative
of more or less stable spermatogenesis (Schulte-Hostedde
and Montgomerie 2006) we also tested the correlation
between intra-ejaculate variance (SD) in sperm length and
body measures and condition.
Methods
The study was conducted on a sand martin colony
comprising seven sub-colonies situated on the Tisza River,
Hungary (488 11?N, 218 28?E; Szép 1995) from 27 April to
19 May 2003. Five females found dead at the bottom of the
riverbank were stuffed, fitted with a false cloaca and fixed at
the end of a fishing rod in copulatory posture to serve as
dummies, a method previously used in this species by
Nicholls and collaborators (2001). The dummy female was
placed in the centre of a 33 25 cm clapnet (Moudrý,
Czech Republic) that was triggered from the distance to
capture copulating males immediately after they ejaculated.
Dummy females were presented either from 06.15 to
10.50, or from 15.30 to 19.50 (local time), when male sand
martins exhibit the highest probability of copulating with a
female mount (Nicholls 2000). The five models were used
alternatively and presented for 30 min or until a male
copulated with it. If no male copulated with the dummy
female, we moved to another sub-colony. If a male
copulated with the dummy, we collected the ejaculate and
immediately recorded sperm motion. The dummy was
afterwards presented again in the same colony. The time of
copulation and when the sample was videotaped were
recorded to the nearest second in order to calculate the time
elapsed between ejaculation and sperm analysis.
The model’s false cloaca was filled with 5 ml of
Dulbecco’s Modified Eagle Medium (4500 mg glucose/l,
110 mg sodium pyruvate/l, L-glutamine, Sigma Aldrich) to
hydrate the transferred ejaculate and facilitate its collection.
The ejaculate collected from the false cloaca was mixed to
648
995 ml pre-warmed (408C) Dulbecco’s Medium leading to
1ml of sperm/buffer solution. When the ejaculate was
deposited outside the false cloaca we used the 5 ml of buffer
to hydrate and collect the sample. A 7 ml-sample was then
loaded within a prewarmed (408C) MicroCell (50 mm
chamber depth, Conception Technologies, San Diego) and
the slide placed under a microscope equipped with a
20  bright-field objective under a dark-field condition
generated with a Ph2 annular phase ring. Sperm motion
was recorded for 15 min on a DV cassette using a video
camera fitted to the microscope and plugged to a DV
handycam used as a recorder. Electricity for the apparatus
(microscope, heating stage and controller, video camera)
was supplied by a car battery plugged to a 12
230V AC/
DC 300W converter. While videotaping sperm motion, we
weighed the male (90.5 g) and measured its left tarsus
(91.102 mm), left wing (91 mm), left external tail feather
(91 mm), and sternum (91.102 mm). We sometimes failed
to take all measures causing sample sizes to vary.
Video recordings of sperm motion were later analysed
using the Hobson Sperm Tracker System at the Max Planck
Institute for Ornithology, Seewiesen, Germany. For each
sample, we took five measures of straight line velocity (VSL)
using a 60 s sampling interval and a 60 s pause between
intervals. This allowed us to monitor the dynamics of sperm
velocity over a period of several minutes after ejaculation
and thus to derive sperm longevity from statistical models as
the rate at which sperm velocity declines with time (Froman
and Feltmann 2000). The minimum track time was 1.2 s,
which minimized the risk that the tracking system would
count a moving particle as a motile sperm (Froman and
Feltmann 2000). Among the various sperm motility
parameters computed by the Hobson Sperm Tracker, we
chose to use the straight line velocity (VSL) because it has
been previously shown to be a good predictor of male
fertility in several taxa (reviewed in Pizzari et al. 2004)
including birds (Denk et al. 2005), and it provides a
measure of sperm motility that can readily be used to test
the relationship between sperm speed and sperm length.
Ten to 28 (mean9SE: 1992) sperm were measured for
their total length from steady video pictures.
We captured 60 males that copulated with dummy
females. Some males did not ejaculate during the copulation
(n 22, 36.6%). In eight cases (13.3%) the dummy
attracted several males that successively copulated with it,
resulting in a mixture of ejaculates that could not be used.
Eventually, we obtained measurements of sperm length for
28 males and, due to technical problems, video recordings
of motile sperm from 14 males.
The males that were prone to copulate with our
dummies could form a biased subset of the male popula-
tion. If they were, those males should differ in their mean
morphology and should exhibit smaller variance in those
traits compared to the whole male population. We thus
compared mean values and variances of all traits between
our sample (measures taken by ZN and FH) and a larger
sample of males (ranging from 202 to 580 individuals,
measures taken by TS) captured during the intensive
ringing campaign later in the season.
VSL was square-root transformed prior to analyses to
match modelling assumptions. Analyses involving repeated
measures within the same male were conducted with
Generalized Linear Mixed Models using the Restricted
Maximum-Likelihood estimation method (REML-GLMM,
Littell et al. 2006) and assuming normal distribution of the
error. The male’s identity was declared as the random
subject using an unstructured R matrix and intercepts and
slopes were allowed to vary among males. Degrees of
freedom for fixed effects were estimated with the Sat-
terthwaite approximation, which may yield non-integer
numbers. Modelling assumptions were validated by plotting
model residuals against predicted values and by testing
the residuals’ normality. Analyses were conducted using the
SAS, version 9.1. All tests are two-tailed with significance
level set at a 0.05.

Results
Males who copulated with dummy females did not
statistically differ in size from males captured during the
intensive ringing campaign later in the season (all t B1.85,
P 0.065). However, they differed in their body mass
(t 5.50, PB0.001, df 611). Yet, this difference is likely
to be mostly due to a seasonal change in body mass. Our
sample also showed similar variances in all morphological
traits measured (all F B2.09, P 0.15). The males who
copulated with our dummies can thus be considered as a
representative sample of the whole population.
Sperm length showed variation within and among males
(Table 1, Fig. 1). The average time elapsed between male
ejaculation and the start of the 15 min video recording was
9 min 35 s (range: 5 min 39 s to 11 min 17 s). A first
analysis modelling VSL as a function of time elapsed since
ejaculation and using male identity as a random factor
(REML-GLMM) revealed that: 1) VSL significantly de-
clined with time (b9SE 0.01890.007, F1,13 5.70,
P 0.03), and that 2) males differed in their initial sperm
velocity (different intercepts) and in the rate at which their
sperm velocity declined (different slopes). Entering the
males’ mean sperm length in this model resulted in VSL to
be significantly explained by an interaction between sperm
length and time elapsed since ejaculation (F1,11.6 4.81,
P B0.05). In other words, sperm size was correlated with
sperm longevity, with males having the shortest sperm
exhibiting: 1) the highest initial sperm velocity, and 2) the
steepest decline in velocity, whereas males having longer
sperm showed stable intermediate velocity (Fig. 2).
None of the morphological traits we measured correlated
with total sperm length or intra-ejaculate variation in sperm
length (within ejaculate/male standard deviation) (all ½r½
B0.32, P0.10, n 22
28). Body condition (body mass
accounted for tarsus length used as an explanatory variable
in the model) did not covary with sperm length (mass:
F1,18 0.06, P 0.80; tarsus: F1,18 0.02, P 0.89), or
with intra-ejaculate variation (SD) in sperm length (mass:
F1,18 0.01, P 0.92; tarsus: F1,18 0.00, P 0.98).
Figure 1. Within- and among-male variation in total sperm
length. Circles represent individual means (9SE) in total sperm
length based on 10 to 28 spermatozoa per male. Males are
numbered and ranked from small to large average sperm length.
Morphological traits did also not covary with sperm velocity
(REML-GLMM: time since ejaculation: all F 5.90,
P B0.032; morphological traits: all F B3.49, P 0.09),
except for tarsus length which was negatively correlated with
sperm velocity (REML-GLMM: time since ejaculation:
F1,11.7 5.62, P 0.036; tarsus length: F1,11.2 5.31,
P 0.04). Males having small tarsi had sperm with higher
overall velocity (Fig. 3).
Our modest sample may prevent us from detecting
existing relationships between sperm length, variation in
sperm length or sperm velocity and male morphology. This
is reflected in the low power of all analyses (ranging from
0.05 to 0.48), with the exception of the analysis testing for a
correlation between body mass and sperm velocity (0.79).
Thus, we may have been unable to detect existing relation-
ships that would be revealed with a larger sample.
Discussion
Theoretical developments investigating the circumstances
under which sperm competition would select for increased
sperm length have relied on the intuitive assumption that
sperm length is positively associated with sperm swimming
speed (Gomendio and Roldan 1991, Parker 1998), an
assumption that has seldom been tested. We did not find a
direct correlation between sperm length and sperm velocity
or that sperm longevity negatively covaried with sperm
length. We found that short sperm have a higher initial
velocity but a shorter lifespan whereas long sperm have a
lower initial velocity but a higher longevity. This contradicts
the general assumption that sperm size is traded against
sperm longevity (Cardullo and Baltz 1991, Immler and

Table 1. Variation in total sperm length (mm) among samples of 28 male sand martins.

Mean SE Min. Max. CV Within-male repeatabilitya N0 P

109.6 0.5 105.4 114.2 2.2% 0.20 20.7 B0.001

a
Sensu Lessells and Boag (1987).

649

Figure 2. Sperm straight line velocity (VSL, square-root trans-
formed) as a function of the time since ejaculation and the male
mean total sperm length. Data are predicted values from a REML-
GLMM including the male’s identity as a random factor, allowing
intercepts and slopes to vary among males.
Birkhead 2007, Immler et al. 2007). It also contrasts with
previous studies within (LaMunyon and Ward 1998), and
across species (Gomendio and Roldan 1991, Stockley et al.
1997) that found long sperm to have higher maximum
velocity and/or reduced longevity. It is however consistent
with results found in the sea urchin where sperm velocity and
longevity trade off against each other (Levitan 2000). One
explanation why long sperm swim more slowly but live
longer may be that long sperm are slowed down by higher
friction forces in the media due to their larger surface. Yet,
Figure 3. Sperm straight line velocity (VSL, square-root trans-
formed) as a function of the time since ejaculation and the male
tarsus length. Data are predicted values from a REML-GLMM
including the male’s identity as a random factor, allowing
intercepts and slopes to vary among males.
650
their bigger total length may be correlated with a longer
midpiece. In our study, we were not able to measure
midpiece length. Across bird species, a positive allometry
between midpiece length and flagellum length seems to be
the rule: midpiece length increases more per unit of flagellum
length and is positively correlated with total sperm length
(Immler and Birkhead 2007, Immler et al. 2007). However,
within species, midpiece size generally shows no association
with other components of sperm length (Morrow and Gage
2001a, Birkhead et al. 2005, Malo et al. 2006). Nonetheless,
if longer sperm have a higher mitochondrial load per unit of
size, this may translate into higher ATP content allowing
greater longevity (Froman and Kirby 2005).
Assuming that sperm size increases sperm survival,
theory also predicts that, if the timing between insemina-
tion and fertilisation increases, long sperm will be favoured
(Parker 1998). Conversely, if the risk of sperm competition
decreases, i.e. if the time between insemination and
fertilisation decreases, short sperm should be selected
(Parker 1998). Thus males producing small, fast and
short-lived versus long, slow and long-lived sperm may
have different selective advantages depending on the timing
of insemination relative to fertilisation. When fertilisation
occurs shortly after insemination, fast swimming sperm may
be able to outcompete rivals’ sperm in reaching the storage
organs and the ovum (Birkhead et al. 1999). When
fertilisation occurs long after insemination, slow swimming,
long-lived sperm may have the selective advantage of being
passively lost from the female’s sperm storage glands at a
lower rate and therefore be more numerous by the time of
fertilisation (Froman et al. 2002).
Sperm length and sperm velocity showed substantial
variation among males, as found in other animals (Morrow
and Gage 2001a, Gage et al. 2002, Oppliger et al. 2003,
Birkhead et al. 2005, Malo et al. 2006, Schulte-Hostedde
and Montgomerie 2006). However, sperm velocity and
viability and/or longevity have been found to be important
determinants of a male’s sperm competition ability and are
hypothesised to be under directional selection (Holt et al.
1997, Birkhead et al. 1999, Donoghue et al. 1999, Hunter
and Birkhead 2002, Garcı́a-Gonzáles and Simmons 2005).
Additionally, there is good evidence that sperm length has
co-evolved with female reproductive anatomy (Briskie et al.
1997, Miller and Pitnick 2002) and it is hypothesised to be
under stabilizing selection (Calhim et al. 2007). Thus, what
maintains variation in both sperm velocity and size is
currently unclear. A first hypothesis is that sperm length
and sperm velocity are partially maternally inherited (Pizzari
and Birkhead 2002). This hypothesis has recently been
substantiated by studies showing that variations in sperm
design or motility have a partially sex-biased inheritance
(Ward 2000, Morrow and Gage 2001b, Froman et al.
2002, Birkhead et al. 2005, Dowling et al. 2007). However,
although maternal genetic effects contribute to variations in
sperm traits, their heritability also shows a direct paternal
genetic component (Simmons and Kotiaho 2002, Birkhead
et al. 2005, Simmons and Roberts 2005, Dowling et al.
2007).
A second hypothesis is that sperm characteristics are
condition-dependent sexually selected traits (Rowe and
Houle 1996). Condition-dependence has indeed been
found in sperm swimming speed and sperm length
(Simmons and Kotiaho 2002, Malo et al. 2005, Schulte-
Hostedde and Montgomerie 2006, Garcı́a-Gonzáles and
Simmons 2007). However, we found no significant
correlations between body condition and sperm character-
istics, and even found that males with shorter tarsi produced
sperm with higher overall velocity (Fig. 3).
This latter result is in agreement with another hypoth-
esis that trade-offs exist between sperm traits that
determine a male’s sperm competitive ability and some
other fitness related traits, which may promote variation in
sperm traits. Theory (Parker 1998) predicts that males
differing in their access to fertile females should invest
differently in their ejaculate expenditure with disfavoured
males investing more. Parker (1998) reviewed some
empirical examples supporting this hypothesis. A more
recent example is provided by the feral fowl where
dominant males monopolise fertile females but produce
less mobile sperm compared to subdominant males (Fro-
man et al. 2002, Pizzari et al. 2007). Our results suggest
that such a trade-off might exist in sand martins. Small
males may have less mating opportunities and thus invest
more in sperm competitive ability by producing faster
swimming sperm. Such a trade-off would participate in
maintaining variation in sperm velocity and length. The
lesser access of small males to females however remains to
be investigated.
In summary, we found that total length of sand
martins’ sperm is repeatable within a male’s ejaculate
and shows substantial variation between males. Sperm
length was associated with sperm velocity: males with
shorter sperm have sperm that swim initially faster but die
sooner. This result contradicts the intuitive idea that long
sperm swim faster. Such a trade-off between maximum
velocity and longevity among sperm of different sizes may
promote different but equally successful insemination
strategies. Additionally, small males produced sperm with
higher average velocity. If smaller males have a lower
access to females this may be a strategy to compensate for
their lower mating opportunities. Our sample is modest
and further studies are welcome to confirm our results.
Studies on a variety of species where male phenotype
preconditions access to fertile females are also needed to
test whether males develop alternative fertilisation strate-
gies by producing sperm of different length, velocity and/
or longevity.
Acknowledgements
We are especially grateful to Tim Birkhead for
constructive discussion in planning the project. We also thank
Simone Immler and two anonymous referees for their valuable
comments on earlier drafts of the manuscript and Angelika Denk
for her advice in using the Hobson Sperm Tracker system. We
thank the local chapter of the MME/BirdLife Hungary for
providing field infrastructure for our work. This work was
financially supported by a grant from the Fyssen Foundation to
FH and a grant (OTKA T042879) to TS.
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